Vision Research 42 (2002) 151–157 www.elsevier.com/locate/visres Rat retinal ganglion cells co-express brain derived neurotrophic factor (BDNF) and its receptor TrkB
nica Garc
ıa a, Luis Martinez-Mill
an c, Elena Vecino a,b,*, David Garc
ıa-Grespo a, Mo Sansar C. Sharma d, Eliseo Carrascal b a Departamento de Biolog
ıa Celular e Histolog
ıa, Facultad de Medicina, Universidad del Pa
ıs Vasco, E-48940 Leioa, Vizcaya, Spain Departamento de Anatom
ıa e Histolog
ıa Humana, Facultad de Medicina, Universidad de Salamanca, E-37007 Salamanca, Spain c Departamento de Neurociencias, Facultad de Medicina, Universidad del Pa
ıs Vasco, Leioa E-48940 Leioa, Vizcaya, Spain d Department of Ophthalmology, New York Medical College, Valhalla, NY 10595, USA b Received 29 March 2001; received in revised form 6 August 2001 Abstract The expression of brain derived neurotrophic factor (BDNF) and its preferred receptor (TrkB) in rat retinal ganglion cells (RGCs) have been determined in the present study. To identify RGCs retrograde labelling was performed with fluorogold (FG). Subsequently, retinas were immunostained with antibodies to BDNF and TrkB. We found that all RGCs labelled with FG express both BDNF and its preferred receptor, TrkB. Moreover, displaced amacrine cells were also found to be immunolabelled by both antibodies. Thus BDNF/TrkB signalling in RGCs probably involves endogenous BDNF produced by the RGCs themselves. Ó 2002 Elsevier Science Ltd. All rights reserved. Keywords: Brain derived neurotrophic factor; TrkB; Ganglion cell; Retina; Fluorogold; Neurotrophin; Receptor 1. Introduction Brain derived neurotrophic factor (BDNF) and its receptor TrkB (Middlemas, Lindberg, & Hunter, 1991) have been proposed to play an important role in the neuroprotection of retinal ganglion cells (RGCs). Recent studies have shown that exogenously applied BDNF promotes the survival and prevents the death of RGCs both in vivo, after axotomy (Mansour-Robaey, Bray, & Aguayo, 1992; Mey & Thanos, 1993; ManosurRobaey et al., 1994; Peinado-Ram
on, Salvador, Villegas-P
erez, & Vidal-Sanz, 1996), and in vitro (Johnson, Barde, Schwab, & Thoenen, 1986; Thanos, B€ arh, Barde, & Vanselow, 1989). Moreover, exogenously applied BDNF enhances optic axon branching in vivo (CohenCory & Fraser, 1995) and protects RGCs from ischemic injury (Unoki & La Vail, 1994). Although these studies suggest that exogenously applied BDNF can play an * Corresponding author. Address: Departamento de Biolog
ıa Celular e Histolog
ıa, Facultad de Medicina, Universidad del Pa
ıs Vasco, Leioa E-48940, Vizcaya, Spain. Tel.: +34-94-464-7700; fax: +34-94464-8966. E-mail address: gcpvecoe@lg.ehu.es (E. Vecino). important role in RGC survival, it is presently uncertain if endogenous BDNF can also mediate neuroprotection (Gao, Qiao, Hefti, Hollyfield, & Knusel, 1997; Vecino, Ugarte, Nash, & Osborne, 1999). Nevertheless the endogenous levels of BDNF mRNA and protein in the retina have been shown to be modulated by injury to the optic nerve (Gao et al., 1997), by retinal ischemia, (Vecino, Caminos, Ugarte, Mart
ın-Zanca, & Osborne, 1998) and by injection of NMDA into the eye (Vecino et al., 1999), suggesting that it may play some relevant role following visual system injury. The presence of BDNF in the RGC layer of the retina was first shown at the level of mRNA, by in situ hybridisation (Qiao, Gao, & Hollyfield, 1994) and later at the level of protein synthesis (Vecino et al., 1998). Moreover, the RGC layer of the retina also contains cells which express the BDNF-preferring receptor TrkB, at both the mRNA and protein level (Jelsma, Friedman, Berkelaar, Bray, & Aguayo, 1993; Vecino et al., 1998). BDNF action in the RGC may involve the activation of the BDNF/TrkB ligand/receptor complex at nerve terminals, and its subsequent internalisation and retrograde transport to the cell body. Indeed, interruption of this retrograde transport of BDNF and TrkB in the 0042-6989/02/$ - see front matter Ó 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 4 2 - 6 9 8 9 ( 0 1 ) 0 0 2 5 1 - 6 152 E. Vecino et al. / Vision Research 42 (2002) 151–157 optic nerve head may contribute to the damage observed in acute and chronic glaucoma (Pease, McKinnon, Quigley, Kerrigan-Baumrind, & Zack, 2000). Although neurotrophins in the retina have been extensively studied, at the level of the developmental and functional roles of these molecules, a precise description of the distribution of BDNF and its high affinity receptor TrkB in the RGC layer of the retina is still lacking. The RGC layer contains ganglion cells, glial cells and other cell types. It is estimated that approximately 50% of neurons in the ganglion cell layer of adult rats are displaced amacrine cells (Perry, 1981). Thus, the purpose of this study was to identify the population of RGCs in the rat retina which express both BDNF and the TrkB receptor. The most suitable method currently used for identifying the complete RGC population consists of labelling these cells from their target in the brain using tracers, such as fluorogold (FG), which are retrogradely transported. Once the whole population of RGCs were thus labelled, we immunostained retinas with antibodies to BDNF or TrkB, in order to identify the population of RGCs that express BDNF or TrkB. The presence of double-labelled cells FG/BDNF or FG/TrkB was determined in both sectioned and wholemounted retinas. We clearly distinguished three different sizes (small, medium and large) of RGCs in retrogradely labelled, wholemounted retinas. The morphology of these cell groups largely corresponded to the alpha, beta and gamma ganglion cell types. Moreover, we found that all cells in the ganglion cell layer which were labelled with FG also expressed BDNF and TrkB, indicating coexpression of the ligand and its receptor in RGCs. In addition, displaced RGCs observed in the inner nuclear layer (INL), which constitute less than 1.5% of the total population of RGCs, were also labelled with antibodies to both the neurotrophin and its receptor. Vidal-Sanz, Bray, & Aguayo, 1988; Villegas-P
erez, Vidal-Sanz, Rasminsky, Bray, & Aguayo, 1993). Rats were anaesthetised and the midbrain exposed. A small piece of gelatine sponge (Sponhongostan Film, Ferronsan, Denmark) soaked in 0.9% NaCl containing 3% FG and 10% dimethylsulfoxide was laid over the superior colliculi and lateral geniculate nuclei, both of which are the targets of RGC axon projections. The animals were allowed to recover and six days later were killed with an overdose of anaesthetic and perfused through the ascending aorta with 0.9% NaCl followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). Eyes were enucleated and the retinas were dissected, prepared as wholemounts, postfixed for an additional hour, and mounted, vitreal side up, on gelatine coated slides. The ganglion cell layer was examined using fluorescence microscopy with an ultraviolet filter set. Once the retinas were examined and the retrograde transport to RGCs was confirmed, some were prepared for wholemount immunostaining with antisera to BDNF or TrkB, while others were cryoprotected in 30% sucrose in PBS, embedded in Tissue Tek (Leika) and sectioned at 14 lm in a cryostat. 2.2. Retinal ganglion cell counts Mean densities of FG-labelled RGCs in the ganglion cell layer of the retina were estimated following previously described methods (Villegas-P
erez et al., 1993; Laquis, Chaudhary, & Sharma, 1998). Briefly, labelled RGCs were counted by the same observer from photographs of 12 rectangular (0:36  0:24 mm2 ) areas of each retina, three in each quadrant (superotemporal, inferotemporal, superonasal, and inferonasal) at distances of 1, 2 and 3 mm from the optic disc. The number of labelled cells in the 12 photographs was divided by the area of the region and pooled to calculate mean densities of labelled neurons/mm2 for each retina. 2. Material and methods 2.3. Immunohistochemical procedure 2.1. Retrograde labelling of retinal ganglion cells Sectioned and wholemounted retinas were rinsed in PBS containing 0.25% Triton X-100 (PBST), incubated for 1 h at room temperature (RT) with blocking solution containing PBST and 1% bovine serum albumin (BSA; fraction V, Sigma). Sections were incubated overnight at 4°C with rabbit anti-BDNF (diluted to 1:200; Santa Cruz #546) or rabbit anti-TrkB (diluted 1:100; Transduction Laboratories #119) antisera. The wholemounted retinas were incubated with antisera at the same dilution, but with constant agitation at 4°C for 48 h. After rinsing in PBST, the retinas were incubated in goat anti-rabbit IgG conjugated to Texas-red (diluted 1:200; Molecular Probes). Sections were thus incubated for 1 h whereas the whole free-floating retinas were incubated with secondary antibody for 4 h. Sections and wholemounts Eleven adult Sprague–Dawley rats (each weighing 225–250 g) were used in the present study. Rats were housed in standard cages, fed ad libitum and maintained in temperature-controlled rooms with a 12 h light–12 h dark cycle. For all experimental manipulations, the animals were anaesthetised with intraperitoneal injections of 7% chloral hydrate (0.42 mg/g body weight). Experiments were carried out in accordance with the European Union guidelines and the ARVO Statement for the use of Animals in Ophthalmic and Vision Research. To identify RGCs, we labelled them with the fluorescent tracer FG (Fluorochrome, Englewook CO) following previously described techniques (Villegas-P
erez, E. Vecino et al. / Vision Research 42 (2002) 151–157 were rinsed in PBS and then coverslipped with PBS/ glycerol (1:1) and examined by epifluorescence microscopy. After initial microscopic examination, some wholemounted retinas were cryoprotected and sectioned. 2.4. Immunohistochemical controls Labelling specificity was assessed by (I) omission of the primary antiserum, replacing it with PBST–BSA, (II) omission of secondary antibody, (III) preadsorption of primary antibodies with their respective antigenic peptides (2–10 lg/ll of peptide) and (IV) heterologous preadsorption, preadsorbing the anti-BDNF antiserum with the TrkB peptide and the anti-TrkB antiserum with the BDNF peptide. 3. Results FG fluorescence was found in the cytoplasm of RGC somata and occasionally in the proximal dendrites of these cells. Intense, homogenous FG labelling was observed, in addition to a more punctate-type distribution of label. Six days after FG application, the mean density of the FG-labelled RGCs/mm2 was calculated to be 2420  50 (mean  SEM). This mean density is comparable to estimates of rat RGC densities determined with the same tracer or with other retrogradely transported fluorescent tracers, similarly applied to the retino-recipient targets (Villegas-P
erez et al., 1988; Peinado-Ram
on et al., 1996). The specificity of the antisera was demonstrated with the controls which gave negative results in control cases I, II and III, while in case IV, immunostaining was unaltered, demonstrating that preadsorption under these conditions was specific. Moreover, the specificity of these antisera in the fish and rat retina has already been reported (Vecino et al., 1998; Caminos, Becker, MartinZanca, & Vecino, 1999). In both the sectioned and wholemounted retinas, all RGCs labelled with FG were also immunostained by the antisera to BDNF (Figs. 1A–D and 2) and to TrkB (Fig. 1E and F). Thus we can conclude that in the retina of the rat, both BDNF and its preferred receptor TrkB are expressed in all RGCs (Fig. 1A and B). A number of cells located in the RGC layer were BDNF immunopositive but were not FG immunolabelled. These cells were presumably displaced amacrine cells (Figs. 1A, B, and 2) BDNF and TrkB were also present in FG-positive cells located in the INL. These cells were evidently displaced RGCs. We found that BDNF and TrkB immunolabelled cells could be classified into at least three groups, on the basis of their size (<15 lm, 15–25 lm and larger than 25 lm; Fig. 2C and D). In addition, FG, BDNF and TrkB 153 immunolabelling was observed to be differentially located within the cell body. Thus, while FG was located in the perinuclear area of the soma, BDNF and TrkB immunoreactivity was located in more peripheral cytoplasm and/or in the cytoplasmic membrane (Figs. 1 and 2). This differential distribution could be more clearly observed when the corresponding images were superposed using the Adobe PhotoShop program (Fig. 3). 4. Discussion In the present study we have shown that all, or at least the vast majority of RGCs are retrogradely labelled with FG and that they contain both BDNF and TrkB immunoreactivity. These results, together with previous in situ hybridisation experiments which demonstrated the presence of BDNF and TrkB mRNAs in the RGC layer, raise the possibility that locally produced BDNF may play an important role in the activation of RGC TrkB receptors in addition to the retrogradely transported ligand. It is known that following optic nerve axotomy, cell death can occur (Villegas-P
erez et al., 1993) by apoptosis (Berkelaar, Clarke, Wang, Bray, & Aguayo, 1994; Garc
ıa-Valenzuela, Gorczyca, Darzynkiewicz, & Sharma, 1994). It has recently been shown that different RGC types have different survival responses to injury and regeneration (Thanos & Mey, 1995) with the large alpha RGCs being more vulnerable to injury. Intravitreal injection of BDNF and other neurotrophic factors favours the survival of RGCs after optic nerve axotomy (Mansour-Robaey et al., 1992; Mey & Thanos, 1993; Mansour-Robaey, Clarke, Wang, Bray, & Aguayo, 1994; Peinado-Ram
on et al., 1996). In the present study, we found that all RGCs express both BDNF and its receptor, indicating that the differential responses of RGCs to axotomy do not depend on the presence or absence of BDNF or TrkB. Rather, RGC survival may depend on there being a sufficient level of expression of BDNF and its receptor in damaged cells post axotomy. The observation that injection of BDNF into the eye can lead to RGC rescue following trauma, supports this hypothesis. Retinal ischemia produces an elevation of extracellular levels of glutamate in the retina, an interruption of retrograde transport in the optic nerve and an obstruction of the arrival at the cell body of molecules required for the survival of RGCs (Pease et al., 2000). Even when this alteration in retrograde transport is present, retinal ischemia induces an increase of BDNF protein synthesis in RGCs, at least during the first few hours after damage, corroborating the idea of locally produced vs. retrogradely transported BDNF. This increased synthesis of BDNF may represent an endogenous neuroprotective response by the RGCs (Vecino et al., 1998, 1999). 154 E. Vecino et al. / Vision Research 42 (2002) 151–157 Fig. 1. Immunohistochemical distribution of BDNF, TrkB and FG in the rat retina. A and B illustrate BDNF-immunolabelled and FG-labelled RGCs, respectively. The arrows point to a displaced RGC which presents both BDNF and FG. The asterisks indicate two displaced amacrine cells which are not labelled with FG but are BDNF immunopositive. Scale bar, 25 lm. C and D are low-magnification photomicrographs of BDNF- and FG-stained cells respectively. Note that in the INL, there are many BDNF immunoreactive cells. Scale bar, 25 lm. E and F are TrkB and FG-stained cells respectively. The cytoplasmic nature of TrkB staining in RGCs is apparent. Scale bar, 25 lm. GCL (ganglion cell layer), IPL (inner plexiform layer), INL (inner nuclear layer). However, after this initial response there is a decrease in the expression of BDNF in RGCs which could be due to an alteration in the metabolism of the affected cells or also to an interruption of the retrograde transport from their target areas in the brain where neurons expressing BDNF mRNA have been described (Wetmore, Ernfors, Persson, & Olson, 1990; Friedman, Olson, & Persson, 1991; Conner, Lauterborn, Yang, Gall, & Varson, 1997). Recent experiments have also demonstrated an accumulation of TrkB in the optic nerve head after retinal ischemia, possibly due to an interruption of the retrograde transport of the receptor (Pease et al., 2000). It thus seems likely that both BDNF and TrkB can be retrogradely transported to the rat retina from other areas of the brain. However, as these authors failed to find TrkB in RGCs, it is possible that local, endogenous synthesis may represent the principal source of TrkB receptor in these cells. Nevertheless, in the case of BDNF, anterograde transport from other retinal cells to RGCs cannot be ruled out since the anterograde transport of BDNF has been reported in the central nervous system of the rat (Altar et al., 1997; Conner et al., 1997; Altar & DiStefano, 1998; Fawcett et al., 2000), and chick visual system (von Bartheld et al., 1996; Herzog & von Bartheld, 1998). Little is known about the mechanism by which FG is retrogradely transported in neurons. However it has been demonstrated that all or at least the vast majority of RGCs are retrogradely labelled when FG is applied to the targets of RGC axon projection areas of the brain (Villegas-P
erez et al., 1988, 1993) as in the present study. In contrast, it is well known that BDNF is retrogradely transported in association with TrkB. Recently it has been demonstrated that BDNF is associated with vesicular-like structures in both the cell body and processes of neurons. Differential centrifugation data have also E. Vecino et al. / Vision Research 42 (2002) 151–157 155 Fig. 2. Flat mounted retinas retrogradely labelled with FG (A and C) and immunolabelled with anti-BDNF antibodies (B and D). A and B represent the same area of the retina. BDNF is clearly absent in cell nuclei, corroborating the specificity of the immunolabelling. Note that there are more BDNF labelled RGCs (B) than FG stained cells (A). Scale bar for both photographs, 25 lm. C and D are high magnifications of the same area of the retina where the three different sizes of RGCs labelled with FG in C and with BDNF in D are well represented. The asterisks indicate cells located in the GCL which are not RGCs but which are BDNF immunoreactive. Scale bar, 25 lm. shown that BDNF is present in microvesicles isolated from a synaptosomal fraction. These data, taken together with the results of the present study are consistent with the idea that BDNF produced in the neuron soma is transported anterogradely, targeted towards the regulated secretory pathway and localised within the presynaptic compartment of neurons as part of a secretory mechanism for BDNF (Fawcett et al., 1997). We observed a perinuclear distribution of FG (Fig. 1B), and a more peripheral cytoplasmic distribution of BDNF and TrkB (Fig. 1A). Further studies at the level of electron microscopy will help us to elucidate the way in which neurons store and transport neurotrophins and other substances like FG. The ubiquitous presence of BDNF immunoreactivity in all size-classes of RGCs contrasts with the observation that only a low percentage of cells in the ganglion cell layer express BDNF mRNA after axotomy (Gao et al., 1997). Nevertheless, these differences are quite likely due to alterations in mRNA synthesis following axotomy. In the present study, we have shown that both BDNF and its preferred receptor TrkB are expressed by the majority of rat RGCs, raising the possibility that BDNF/ TrkB signalling in rat RGCs may occur through autocrine/paracrine mechanisms. Our results indicate that RGC vulnerability following injury is not due to the absence of the receptor ligand complex but may be due 156 E. Vecino et al. / Vision Research 42 (2002) 151–157 Fig. 3. Adobe PhotoShop series of images from the RGC layer (A–F). Using the Murphing program, it is possible to separate the co-localisation of FG (green) and BDNF (red) located in the RGCs into a series of images. Note that D shows the co-localisation of both substances in most cells. The arrow in D points to a cell in which it is possible to distinguish clearly the peripheral location of BDNF around the more central location of FG. The arrowhead in F points to a cell which expresses BDNF but does not contain FG. to insufficient levels of expression of these molecules. These findings of BDNF/TrkB expression on all RGCs, including the subpopulation of displaced RGCs (1.5% of the total population; Thanos, 1988), contribute to a better understanding of RGCs which will be essential in order to develop future clinical neuroprotective treatments. Acknowledgements We wish to thank Dr. Peter Hitchcock for his suggestions, comments and revision of the manuscript, and the agency ACTS (acts@euskalnet.net) for revising the english of our paper. This work was supported by the grants to E.V. from the MEC (PM 97-0047), the E. Vecino et al. / Vision Research 42 (2002) 151–157 Gobierno Vasco (PI-1998-81), Universidad del Pa
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